Eighty percent of smokers who quit relapse within a year. Although there are many reasons for relapse, the active ingredient in cigarettes, nicotine, has properties of an addictive substance. All of the actions of nicotine are not known, but nicotine is known to act as receptor sites in nervous tissues. Its action at acetylcholine receptor sites may be related to its addictive properties. The nicotinic acetylcholine receptors in mammalian brain have not been well-characterized. Nicotine binds with high affinity (Kd 1-10 nM) to at least one population of brain nicotinic receptors. The high-affinity binding to brain tissue is a paradox. Electrophysiological studies suggest that micromolar concentrations of nicotine are necessary for receptor activation. It has been suggested that the brain receptors are desensitized during the assay procedure (in which 10-20 nM concentrations of nicotine are used), indicating a 1000-fold difference between the K-activation (K- act) and K-desensitization (K-des) of nicotinic receptors in mammalian brain. Previous work in our laboratory indicates that the K-act and K-des of PC12 cells, which possess a well-characterized neuronal receptor, are 7.7 X 10(5) M and 8.2 X 10(5) M, respectively. On the basis of these data, we question whether the K-des and K-act in brain tissue would differ by 3 orders of magnitude. This question cannot be resolved using brain tissue in vivo. We therefore propose to study this question in primary neuronal brain cultures using 22Na+ influx. 22Na+ influx has not previously been demonstrated in primary neuronal cultures derived from mammalian brain. Therefore, the goal of this research is to demonstrate nicotinic receptor mediated 22Na" influx in mammalian brain culture. Experiments using 22Na+ influx and nicotine and cytisine as agonists will be performed to determine the K-act and K-des of the receptors. Influx will be also carried out in the presence of nicotinic antagonists. These experiments will serve as the basis for future inves- tigations of the physiological properties of brain nicotinic receptors.